Urinary tract infections (UTIs) afflict approximately 7 million women in the U.S. annually, causing significant morbidity and costing $3.5 billion for treatment. Most community-acquired UTIs are due to infection by uropathogenic E. coli (UPEC) that elicit an inflammatory response in the bladder during acute cystitis. Identifying novel therapeutic targets against UPEC is critical because of both the high incidence of UTIs and the increasing prevalence of antimicrobial resistance among UPEC isolates. NF:B is a pivotal mediator of inflammation and other innate responses, so we previously examined urothelial NF:B responses to UPEC and demonstrated that UPEC possess NF:B suppressive activity (NSA). To characterize the molecular basis of NSA, we performed a genetic screen and identified NSA mutants in UPEC that enhance urothelial inflammation, increase apoptosis, and exhibited attenuated intracellular proliferation. The NSA mutants are defective for activities of normal surface structure biosynthesis, and the involved surface structures are potential activators of innate host responses through Toll-like receptors (TLRs). TLR signaling includes the I:B kinase complex (IKK), where other receptor signals converge with TLR signals that activate NF:B, such as signals induced by EGF and TNF. Since UPEC also modulates EGF and TNF signaling, we hypothesize that UPEC modulation of innate responses by NSA is mediated by bacterial macromolecules that differentially induce TLR signals impinging upon IKK, thus altering key events of UTI pathogenesis that can be exploited to develop novel therapies. Indeed, NSA mutants modulate I:B phosphorylation, induce elevated urothelial apoptosis, are defective for intracellular proliferation, and stimulate enhanced protection in a UTI vaccine assay. In Aim 1, we will identify the UPEC molecules(s) mediating NSA. In Aim 2, we will identify the urothelial targets of UPEC NSA. In Aim 3, we will quantify the impact of NSA on UPEC-induced apoptosis and UPEC invasion and intracellular proliferation. In Aim 4, we will determine the therapeutic potential of UPEC NSA mutants as live vaccines. For all aims, we will employ UPEC strain NU14 and isogenic mutants that differentially induce host responses, and we will use both human cell culture and murine UTI models. These studies lay the groundwork for development and optimization of novel and effective live attenuated vaccines for UTI.